Thiazoline acid derivatives

ABSTRACT

The present invention relates to novel thiazoline acids and derivatives thereof useful as chelators of trivalent metals in therapeutic applications. For example, the thiazoline acid derivatives are useful in diagnosing and treating pathological conditions associated with an excess of trivalent metals in humans and animals.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/300,071, filed Nov. 20, 2002, which is a continuation of U.S.application Ser. No. 09/531,753, filed Mar. 20, 2000, and issued on May6, 2003, as U.S. Pat. No. 6,559,315, which is a continuation of U.S.application Ser. No. 09/144,103, filed Aug. 31, 1998, and issued on Jul.4, 2000 as U.S. Pat. No. 6,083,966. The entire teachings of the aboveapplications are incorporated herein by reference.

GOVERNMENT SUPPORT

Research leading to the completion of the invention was supported inpart by Grant Nos. 3203522-12, RO1HL42817 and RO1DK49108 awarded by theNational Institutes of Health (NIH). The United States Government hascertain rights in and to the claimed invention.

BACKGROUND OF THE INVENTION

While many organisms are auxotrophic for Fe(III), because of theinsolubility of the hydroxide (K_(sp)=1×10⁻³⁸) [Acc. Chem. Res., Vol.12, Raymond et al., “Coordination Chemistry and Microbial IronTransport,” pages 183-190 (1979)] formed under physiological conditions,nature has developed rather sophisticated iron storage and transportsystems. Micro-organisms utilize low molecular weight ligands,siderophores, while eukaryotes tend to utilize proteins to transportiron, e.g. transferrin, and store iron, e.g., ferritin [Trends inBiochem. Sci., Vol. 11, Bergeron, “Iron: A Controlling Nutrient inProliferative Processes,” pages 133-136 (1986)].

Iron metabolism in primates is characterized by a highly efficientrecycling process with no specific mechanism for eliminating thistransition metal [Clin. Physiol. Biochem., Vol. 4, Finch et al, “IronMetabolism,” pages 5-10 (1986); Ann. Rev. Nutri., Vol. 1, Hallberg,“Bioavailability of Dietary Iron in Man,” pages 123-147 (1981); N. Engl.J. Med., Vol. 306, Finch et al, “Perspectives in Iron Metabolism,” pages1520-1528 (1982); and Medicine (Baltimore), Vol. 49, Finch et al,“Ferrokinetics in Man,” pages 17-53 (1970)]. Because it cannot beeffectively cleared, the introduction of “excess iron” into this closedmetabolic loop leads to chronic overload and ultimately to peroxidativetissue damage [The Molecular Basis of Blood Diseases, Seligman et al,“Molecular Mechanisms of Iron Metabolism,” page 219 (1987); Biochem. J.,Vol. 229, O'Connell et al, “The Role of Iron in Ferritin- andHaemosiderin-Mediated Lipid Peroxidation in Liposomes,” pages 135-139(1985); and J. Biol. Chem., Vol. 260, Thomas et al., “Ferritin andSuperoxide-Dependent Lipid Peroxidation,” pages 3275-3280 (1985)]. Thereare a number of scenarios which can account for “iron overload,” e.g.,high-iron diet, acute iron ingestion or malabsorption of the metal. Ineach of these situations, the patient can be treated by phlebotomy [Med.Clin. N. Am., Vol. 50, Weintraub et al, “The Treatment ofHemochromatosis by Phlebotomy,” pages 1579-1590 (1966)). However, thereare iron-overload syndromes secondary to chronic transfusion therapy,e.g., aplastic anemia and thalassemia, in which phlebotomy is not anoption [Iron in Biochemistry and Medicine, Vol. II, Hoffbrand,“Transfusion Siderosis and Chelation Therapy,” page 499 (London, 1980)].The patient cannot be bled, as the origin of the excess iron is thetransfused red blood cells; thus, the only alternative is chelationtherapy. However, to be therapeutically effective, a chelator must beable to remove a minimum of between 0.25 and 0.40 mg of Fe/kg per day[Semin. Hematol., Vol. 27, Brittenham, “Pyridoxal IsonicotinoylHydrazone: An Effective Iron-Chelator After Oral Administration,” pages112-116 (1990)].

Although considerable effort has been invested in the development of newtherapeutics for managing thalassemia, the subcutaneous (sc) infusion ofdesferrioxamine B, a hexacoordinate hydroxamate iron chelator producedby Streptomyces pilosus [Helv. Chim. Acta, Vol. 43, Bickel et al,“Metabolic Properties of Actinomycetes. Ferrioxamine B,” pages 2129-2138(1960)], is still the protocol of choice. Although the drug's efficacyand long-term tolerability are well-documented, it suffers from a numberof shortcomings associated with low efficiency and marginal oralactivity.

Although a substantial number of synthetic iron chelators have beenstudied in recent years as potential orally active therapeutics, e.g.,pyridoxy]isonicotinoyl hydrazone (PIH) [FEBS Lett., Vol. 97, Ponka etal, “Mobilization of Iron from Reticulocytes: identification ofPyridoxal isonicotinoyl Hydrazone as a New Iron Chelating Agent,” pages317-321 (1979)], hydroxypyridones [J. Med. Chem., Vol. 36, Uhlir et al,“Specific Sequestering Agents for the Actinides. 21. Synthesis andInitial Biological Testing of Octadentate MixedCatecholate-hydroxypyridinonate Ligands,” pages 504-509 (1993); andLancet, Vol. 1, Kontoghiorghes et al,“1,2-Dimethyl-3-hydroxypyrid-4-one, an Orally Active Chelator for theTreatment of Iron Overload,” pages 1294-1295 (1987)] andbis(o-hydroxybehzyl)-ethylenediaminediacetic acid (HBED) analogues [Ann.N.Y. Acad. Sci., Vol. 612, Grady et al, “HBED: A Potential Oral IronChelator,” pages 361-368 (1990)], none has yet proven to be completelysatisfactory. Interestingly, the siderophores have remained relativelyuntouched in this search. Their evaluation as iron-clearing agents hasnot at all paralleled the rate of their isolation and structuralelucidation. In fact, until recently, beyond DFO, only two of some 100siderophores identified have been studied in animal models: enterobactin[Gen. Pharmac., Vol. 9, Guterman et al, “Feasibility of Enterocheiin asan Iron-Chelating Drug: Studies with Human Serum and a Mouse ModelSystem,” pages 123-127 (1978)] and rhodotorulic acid [J. Pharmacol. Exp.Ther., Vol. 209, Grady et al, “Rhodotorulic Acid-investigation of itsPotential as an Iron-Chelating Drug,” pages 342-348 (1979)]. While theformer was only marginally effective at clearing iron, the lattercompound was reasonably active. Unfortunately, both of these cyclicsiderophores exhibited unacceptable toxicity, and neither possessed anyoral activity. They were abandoned as there were any number of syntheticchelators with equally unsatisfactory properties from which to choose.

U.S. patent application Ser. No. 08/624,289 filed March 29, 1996, theentire contents and disclosure of which are incorporated herein byreference, discloses certain 2-pyridyl-Δ²-thiazoline-4-carboxylic acidsand derivatives thereof useful for the treatment of human and non-humananimals in need of therapy entailing the prevention of deposition oftrivalent metals and compounds thereof in their tissues, as well as theelimination of such metals and compounds from biological systemsoverloaded therewith.

It is an object of the present invention to provide additional novelthiazoline acids and derivatives thereofwhich, because of differentvolumes of distribution in patients and different lipophilicities thanthe derivatives of the prior art, provide the ability to control thepharmacokinetic properties and toxicities of the drugs.

Another object of the present invention is to provide novelpharmaceutical compositions for and methods of treatment of human andnon-human animals in need of therapy entailing the prevention ofdeposition of trivalent metals and compounds thereof in tissues thereof,as well as the elimination of such metals and compounds from systemsoverloaded therewith.

SUMMARY OF THE INVENTION

The above and other objects are realized by the present invention, oneembodiment of which comprises compounds of the formula:

wherein: Z is CH or N;

-   -   R is H or acyl;    -   R₁, R₂, R₃ and R₅ may be the same or different and represent H,        alkyl or hydrocarbyl arylalkyl having up to 14 carbon atoms; and    -   R₄ is H or alkyl having 1-4 carbon atoms;        a salt thereof with a pharmaceutically acceptable acid or a        pharmaceutically acceptable complex thereof.

Another embodiment of the invention relates to pharmaceuticalcompositions in unit dosage form comprising a therapeutically effectiveamount of the above compound and a pharmaceutically acceptable carriertherefor.

An additional embodiment of the invention concerns methods of preventingor treating a pathological condition in a human or non-human animal thatis associated with an excess of a trivalent metal, ion or compoundthereof comprising administering to the animal a therapeuticallyeffective amount of the compound defined above.

BRIEF DESCRIPTION OF THE DRAWING

The Figure depicts a reaction scheme for preparing the compounds of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated on the discovery that compounds ofthe above formula are valuable bioactive chelators or sequestrants fortrivalent metals such as Fe, Al and Cr. They can be administered tohuman and non-human mammals to prevent the deposition of, e.g., iron, inthe tissues thereof. They are also useful for the elimination of, e.g.,iron, from such mammals afflicted with, e.g., haemochromatosis,haemosiderosis and also cirrhosis. They also find application indialysis, encephalopathy, osteomalacia and Alzheimer's disease.

The compounds described above are characterized by the asymmetric carbonatom marked with an asterisk (*) The bonds surrounding these carbonatoms are arranged tetrahedrally and the substituents thus bonded to theasymmetric carbon atoms are in fixed positions. The formula representsoptical antipodes exhibiting either the (S) or (R) conformation as shownin. (i) and (ii) below:

In the above formula, R is preferably H, but may also be a suitable acylgroup which is cleavable under physiological conditions to the freehydroxyl compounds and a biologically acceptable acid. Such acyl groupsare known in the art, e.g., the acyl radical of a carbonic acid 20semiester, in particular carbonic acid semi-C₁-C₄-alkyl ester orcarbonic acid semi-oxaalkyl ester in which oxaallkyl has 4-13 chainmembers such as an acyl radical —C(═O)—(O—CH₂—CH₂)_(n)—O-Alk in which nis an integer from 0 to 4 and Alk represents C₁-C₄ alkyl, in particularmethyl or ethyl. Such-acyl groups are, for example, methoxycarbonyl,ethoxycarbonyl or 2-(methoxyethoxy)-ethoxycarbonyl. Further acylradicals are, for example, C₁-C₃-alkanoyl such as acetyl or propionyl,or mono-substituted or di-substituted carbamoyl such as di-C₁-C₄-alkylcarbamoyl, for example, dimethylcarbamoyl or diethylcarbarmoyl, orC₁-C₄-alkoxycarbonyl-C₁-C₄-alkylcarbamoyl, for example,methoxlycarbonylmethylcarbamoyl, ethoxycarbonylmethylcarbamoyl or2-ethoxycarbonylethylcarbamoyl.

R₁, R₂, R₃ and R₅ may be the same or different and may be H, straight orbranched chain alkyl having up to 14 carbon atoms, e.g., methyl, ethyl,propyl and butyl or arylalkyl wherein the aryl portion is hydrocarbyland the alkyl portion is straight or branched chain, the arylalkyl grouphaving up to 14 carbon atoms.

R₄ is H or straight or branched chain alkyl having 1 to 4 carbon atoms,e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl.

Preferred among compounds of the above formula are those of the formula:

wherein: Z, R, R₁, R₂, R₃, R₄ and R₅ have the meanings ascribed above,as well as salts thereof with pharmaceutically acceptable acids andpharmaceutically acceptable complexes thereof.

Particularly preferred are those compounds of the above formula wherein:

-   -   a. Z is CH and R═R₁═R₂═R₃═R₄═R₅═H;    -   b. Z is N and R═R₁═R₂═R₃ R₄═R₅═H;        and most preferably, the optically pure isomers thereof.

It will be understood that salts of the compounds of the above formulawith pharmaceutically acceptable acids also comprise part of the presentinvention. Suitable such acids include hydrochloric, sulfuric orphosphoric acids, as well as methanesulfonic, arginine, lysine, and thelike.

The invention also includes pharmaceutically acceptable salts of thecarboxylic acids of the above formula. Thus, ammonium salts and metalsalts such as the alkali metal and alkaline earth metals salts, e.g.,sodium, potassium, magnesium or calcium salts, as well as divalent metalsalts such as zinc, and salts with suitable organic amines, there cominginto consideration such salt formation especially aliphatic,cycloaliphatic, cycloaliphatic-aliphatic or araliphatic primary,secondary or tertiary mono-, di- or poly-amines, and also heterocyclicbases. Such amines are, for example, lower alkylamines, for example,triethylamine, hydroxy-lower alkylamines, for example,2-hydroxyethylamine, bis-(2-hydroxyethyl)-amine ortris-(2-hydroxyethyl)-amine, basic aliphatic esters of carboxylic acids,for example, 4-aminobenzoic acid 2-diethylaminoethyl ester, loweralkyleneamines, for example, 1-ethylpiperidine, cycloalkylamines, forexample, dicyclohexylamine, or benzylamines, for example,N,N′-dibenzyl-ethylenediamine, also bases of the pyridine type, forexample, pyridine, collidine or quinoline. Further salts includeinternal salts (zwitterionic forms of compounds of the invention),wherein a basic group, for example, the basic nitrogen atom present inthe pyridine ring, is protonated by a hydrogen ion originating from anacid group in the molecule.

Owing to their high solubility and good tolerability, metal ioncomplexes of compounds of the above formulae, especially with suitableparamagnetic and/or radioactive metals, can be used as contrast agentsin diagnostic medicine, for example, X-ray, radionuclide, ultrasoundand/or magnetic resonance diagnostics.

Compounds of the above formulae may be synthesized according to thereaction scheme set forth in The Figure wherein D-cys is D-cysteine or areactive functional derivative thereof.

Free hydroxy groups present in the compounds of the above formulae areoptionally protected by conventional protecting groups. Such protectinggroups protect the hydroxy groups from undesired condensation reactions,substitution reactions and the like. The protecting groups can beintroduced and removed easily, i.e., without undesirable secondaryreactions taking place, for example, by solvolysis or reduction, in amanner known per se. Protecting groups and the methods by which they areintroduced and split off are described, for example, in “ProtectiveGroups in Organic Chemistry,” Plenum Press, London, New York (1973) andalso in “Methoden der organischen Chemie,” Houben-Weyl, 4th edition,Vol. 15/1, Georg Thieme Verlag, Stuttgart (1974).

Suitable hydroxy-protecting groups are, for example, acyl radicals suchas lower alkanoyl optionally substituted, for example, by halogen suchas 2,2-dichloroacetyl, or acyl radicals of carbonic acid semiesters,especially tert.-butoxycarbonyl, optionally substitutedbenzyloxycarbonyl, for example, 4-nitrobenzyloxycarbonyl,ordiphenylmethoxycarbonyl, alkenyloxycarbonyl, for example,allyloxycarbonyl, or 2-halo-loweralkoxycarbonyl such as2,2,2-trichloroethoxycarbonyl, also trityl or formyl, or organic silylradicals, also etherifying groups that can readily be split off such astert.-lower alkyl, for example, tert.-butyl, or 2-oxa- or2-thia-cycloalkyl having 5 or 6 ring atoms, for example, tetrahydrofurylor 2-tetrahydropyranyl or corresponding thia analogues, and alsooptionally substituted 1-phenyl-lower alkyl such as optionallysubstituted benzyl or diphenylmethyl, there coming into consideration assubstituents of the phenyl radicals, for example, halogen such aschlorine, lower alkoxy such as methoxy, and/or nitro.

A reactive functional derivative of a carboxy group (Y) Is, for example,an acid anhydride, an activated ester or an activated amide, cyano, agroup of the formula —C(OR_(a))₃ or —C(═NH)—R_(a) in which R_(a) islower alkyl. Corresponding derivatives are well known in the art.

Of the anhydrides, the mixed anhydrides are especially suitable. Mixedanhydrides are, for example, those with inorganic acids such ashydrohalic acids, i.e., the corresponding acid halides, for example,chlorides or bromides, also with hydrazoic acid, i.e., the correspondingacid azides. Further mixed anhydrides are, for example, those withorganic carboxylic acids such as with lower alkanecarboxylic acidsoptionally substituted, for example, by halogen such as fluorine orchlorine, for example, pivalic acid or trichloroacetic acid, or withsemiesters, especially lower alkyl semiesters of carbonic acid such asthe ethyl or isobutyl semiester of carbonic acid, or with organic,especially aliphatic or aromatic, sulfonic acids, for example,p-toluenesulfonic acid. Of the activated esters, there may be mentioned,for example, esters with vinylogous alcohols (i.e., enols such asvinylogous lower alkenols), or iminomethyl ester halides such asdimethyliminomethyl ester chloride (prepared from the carboxylic acidand, for example, dimethyl-(1-chloroethylidine)-iminium chloride of theformula(CH₃)₂N^(⊕)=C(Cl)CH₃Cl^(⊖, which can be obtained, for example, from N,N-dimethylacetamide and phosgene), or aryl esters such as preferably suitable substituted phenyl esters, for example, phenyl ester substituted by halogen such as chlorine, and/or by nitro, for example,)4-nitrophenyl ester, 2,3-dinitrophenyl ester or2,3,4,5,6-pentachlorophenyl ester, N-hetero-aromatic esters such asN-benztriazole esters, for example, 1-benztriazole ester, orN-diacylimino esters such as N-succinylamino or N-phthalylimino ester.Suitable activated amides are, for example, imidazolides, also1,2,4-triazolides, tetrazolides or 1,2,4-oxadiazolinonides.

A preferred form of this process according to the invention is thereaction of a compound of the nitrile with a cysteine derivative. Thereaction is carried out in an inert solvent such as an aqueous solventat ambient temperature or, preferably, at slightly elevated temperature,for example, at about 50° to 80° C., and preferably under an inert gasatmosphere.

In resulting compounds in which one or more functional (hydroxy) groupsare protected, the latter can be freed, optionally in stages orsimultaneously, in a manner known per se, by means of solvolysis,especially hydrolysis or acidolysis, or in some cases also by means ofcareful reduction. Silyl protecting groups are advantageously split offwith fluorides, for example, tetraethylammonium fluoride.

Salts of compounds of the invention can be manufactured in a mannerknown per se. Thus, salts of compounds having acidic groups can beformed, for example, by treating with metal compounds such as alkalimetal salts of suitable organic carboxylic acids, for example, thesodium salt of α-ethylcaproic acid, or with inorganic alkali metal oralkaline earth metal salts, for example, sodium bicarbonate, or withammonia or a suitable organic amine, preferably stoichiometricquantities or only a small excess of the salt-forming agent being used.Acid addition salts of compounds of the invention are obtained in acustomary manner, for example, by treating with an acid or a suitableanion-exchange reagent. Internal salts of compounds of the invention(zwitterionic forms) can be formed, for example, by neutralizing thecompounds or salts such as acid addition salts, to the isoelectricpoint, for example, with weak bases, or by treating with liquid ionexchangers.

Salts can be converted in a customary manner into the free compounds:metal and ammonium salts can be converted into the free compounds, forexample, by treating with suitable acids, and acid addition salts, forexample, by treating with a suitable basic agent.

The starting materials are available commercially and/or known or can bemanufactured by known processes.

The racemate can be split in a manner known per se, for example, afterconversion of the optical antipodes into diastereoisomers, for example,by reaction with optically active acids or bases.

The pharmacologically acceptable compounds of the present invention canbe used, for example, for the manufacture of pharmaceutical compositionswhich contain an effective amount of the active substance together or inadmixture with inorganic or organic, solid or liquid, pharmaceuticallyacceptable carriers.

The pharmaceutical compositions according to the invention are thosewhich are suitable for enteral, such as oral, administration and forparenteral, such as subcutaneous, administration to warm-bloodedanimals, especially humans, and which contain the pharmacologicallyactive substance on its own or together with a pharmaceuticallyacceptable carrier. The dosage of the active substance depends on thespecies of warm-blooded animal and on the age and individual condition,the illness to be treated and also on the mode of administration.

The novel pharmaceutical preparations contain from approximately 10% toapproximately 95%, and preferably from approximately 20% toapproximately 90%, of the active substance. Pharmaceutical compositionsaccording to the invention can, for example, be in unit dose form, suchas dragées, tablets, capsules, suppositories or ampoules, and containfrom approximately 0.05 g to approximately 10.0 g, and preferably fromapproximately 0.3 g to approximately 1.0 g, of the active ingredient.

The pharmaceutical compositions of the present invention aremanufactured in a manner known per se, for example, by means ofconventional mixing, granulating, confectioning, dissolving orlyophilizing processes. Pharmaceutical compositions for oral use can beobtained by combining the active substance with one or more solidcarriers, if desired, granulating a resulting mixture and processing themixture or granulate, if desired or necessary after the addition ofsuitable adjuncts, to form tablets or dragée cores. In so doing, theycan also be incorporated into plastics carriers which release the activesubstances or allow them to diffuse in controlled amounts.

Suitable carriers are especially fillers such as guars, for example,lactose, saccharose, mannitol or sorbitol, cellulose preparations and/orcalcium phosphates, for example, tricalcium phosphate or calciumhydrogen phosphate, also binders such as starches, for example, corn,wheat, rice or potato starch, gelatine, tragacanth, methylcellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/orpolyvinylpyrrolidone, and/or, if desired, disintegrators such as theabove-mentioned starches, also carboxymethyl starch, cross-linkedpolyvinylpyrrolidone, agar, alginic acid or a salt thereof such assodium alginate. Adjuncts are especially flow-regulating and lubricatingagents, for example, silica, talc, stearic acid or salts thereof such asmagnesium or calcium stearate, and/or polyethylene glycol. Dragée coresare provided with suitable coatings that are, if desired, resistant togastric juice, there being used, inter alia, concentrated sugarsolutions which optionally contain gum arabic, talc,polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide,lacquer solutions in suitable organic solvents or solvent mixtures or,for the manufacture of coatings that are resistant to gastric juice,solutions of suitable cellulose preparations such as acetylcellulosephthalate or hydroxypropylmethylcellulose phthalate. Coloring substancesor pigments can be added to the tablets or dragée coatings, for example,for the purpose of identification or for indicating different doses ofactive substance.

Other orally administrable pharmaceutical compositions are dry-filledcapsules made of gelatin and also soft, sealed capsules made of gelatinand a plasticizer such as glycerol or sorbitol. The dry-filled capsulesmay contain the active ingredient in the form of a granulate, forexample, in admixture with fillers such as corn starch, binders and/orglidants such as talc or magnesium stearate and optionally stabilizers.In soft capsules, the active ingredient is preferably dissolved orsuspended in suitable liquids or wax-like substances such as fatty oils,paraffin oil or polyethylene glycols, it being possible also forstabilizers to be added.

Other forms of oral administration are, for example, syrups prepared ina customary manner that contain the active ingredient in, for example,suspended form and in a concentration of approximately from 5% to 20%,and preferably approximately 10%, or in a similar concentration thatprovides a suitable single dose when administered, for example, inmeasures of 5 or 10 ml. Also suitable are, for example, powdered orliquid concentrates for preparing shakes, for example, in milk. Suchconcentrates can also be packed in single-dose quantities.

Particularly suitable dosage forms for parenteral administration aresterile aqueous solutions of an active ingredient in water-soluble form,for example, a water-soluble salt, or sterile aqueous injectionsuspensions which contain substances increasing the viscosity, forexample, sodium, carboxymethyl cellulose, sorbitol and/or dextran, andoptionally stabilizers. In addition, the active ingredient, with orwithout adjuvants, can also be in lyophilized form and brought intosolution prior to parenteral administration by the addition of suitablesolvents.

The invention also relates to compositions for diagnostic purposes thatcontain a suitable metal complex of a compound of the formula wherein Z,R₁, R₂, R₃, R₄ and R₅ are as previously defined.

The invention also relates to a method of treatment of pathologicalconditions in a mammal, especially human, which as has been describedhereinabove, are associated with an excess of a trivalent metal cationsuch as aluminum or, especially, iron (III), in the body, which methodcomprises administering, preferably orally, a prophylactically ortherapeutically effective amount of a compound of the formula or of apharmaceutically acceptable salt thereof. There are used for thispurpose especially the above-mentioned pharmaceutical compositions, adaily dose of from approximately 50 mg to approximately 10,000 mg, andpreferably from approximately 300 mg to approximately 1,000 mg, of acompound of the present invention being administered to a warm-bloodedanimal of approximately 70 kg body weight. The dosage can beadministered orally in several, for example, three, individual doses.For systemic, e.g., subcutaneous, administration, the more water-solublesalt forms of the compounds of the formula, e.g., the sodium salt, arepreferred, for example, orally, or alternatively, subcutaneously.

The following examples serve to illustrate the invention, but should notbe construed as a limitation thereof. Temperatures are given in degreesCentigrade.

Preparation of Drugs. Drug solutions were prepared in 60% water, 40%Cremophor RH-40.

EXAMPLE 1

2,4-Dihydroxybenzonitrile was prepared according to the method of Marcusin Ber. dtsch. chem. Ces. 1981, 24, 3651, as follows:

A mixture of 2,4-dihydroxybenzaldehyde (5.0 g, 36.2 mmol), sodiumacetate (5.94 g, 72.4 mmol), nitroethane (5.44 g, 72.4 mmol) and glacialacetic acid (10 ml) was refluxed for 6 hours. After cooling, the mixturewas poured onto ice (100 g) and extracted with ethyl acetate (4×50 ml).The combined organic layers were washed with saturated NAHCO₃ until thepH of the aqueous layer remained at 8, dried (Na₂SO₄) and the solventremoved in vacuo. Flash chromatography (SiO₂, cyclohexane:ethylacetate=1:1) afforded 2,4-dihydroxybenzonitrile (2.87 g, 59%) as a paleyellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ6.33 (d, 1 H, J=8.6 Hz), 6.43(s, 1H), 7.37 (d, 1H, J=8.6 Hz), 10.35 (s, 1H), 10.78 (s, 1H). IR (KBr)2200 cm⁻¹.

EXAMPLE 2

4,5-Dihydro-2-(2,4-dihydroxyphenyl)-thiazole-4(S)-carboxylic acid wasprepared as follows:

D-cysteine hydrochloride monohydrate (6.8 g, 38.7 mmol) was added to asolution of 2,4-dihydroxybenzonitrile (3.5 g, 25.9 mmol) prepared inExample 1, in a mixture of degassed methanol (105 ml) and 0.1 Mphosphate buffer, pH 5.95 (70 ml). NaHCO₃ (3.25 g, 38.7 mmol) wascarefully added and the mixture was stirred at 70° C. under Ar for 54hours. Volatile components were removed under reduced pressure and thesolution was acidified with 1 N HCl to pH 2. The resulting brownprecipitate was vacuum filtered and the solid was washed with water (40ml) and ethanol (20 ml). The crude product was dissolved in saturatedNaHCO₃ (700 ml) and the aqueous solution washed with ethyl acetate(2×200 ml). The aqueous layer was filtered through a fine frit andacidified with 1 N HCl to pH 2. The precipitated product was vacuumfiltered. The aqueous layer was extracted with ethyl acetate (4×400 ml),the combined organic extracts were dried (Na₂SO₄) and the solvent wasremoved in vacuo. The remaining solid was combined with the precipitatedproduct and dried under high vacuum at 40° C. for 12 hours to give4,5-dihydro-2-(2,4-dihydroxyphenyl)-thiazole-4(S)-carboxylic acid (4.08g, 66%), mp 266-268° C. (dec) [Ind. J. Chem., Vol. 15B, Kishore et at,pages 255-257 (1977) for (L)-isomer: 261-262° C.]. ¹H NMR (300 MHz,DMSO-d₆) δ 3.61 (m, 2H), 5.38 (dd, 1H, J=7.2/9.4 Hz), 6.31 (d, 1H, J=2.3Hz), 6.38 (dd, 1H, J=2.3/8.6 Hz), 7.25 (d, 1H, J=8.6 Hz), 10.25 (br s,1H), 12.60 (br s, 1H), 13.15 (br s, 1H). Anal. calc. for C₁₀H₉NO₄S: C50.20, H 3.79, N 5.85. Found: C, 50.13; H, 3.82; N, 5.85.

Compounds of the invention in the scheme of The Figure wherein Z is Nmay be prepared as described above in Examples 1 and 2 substituting thecorresponding pyridyl aldehyde for 2,4-dihydroxybenzaldehyde.

The biological activity and properties of the compounds of the inventionwere evaluated as follows employing4,5-dihydro-2-(2,4-dihydroxyphenyl)-thiazole-4(S)-carboxylic acid (1).

EXAMPLE 3 In Rats

Initial testing of 1 was performed in the non-iron-over-loaded, bileduct-cannulated rat [J. Med. Chem., Vol. 34, Bergeron et al, “Synthesisand Biological Evaluation of Hydroxamate-Based Iron Chelators,” pages3182-3187 (1991)]. The drug was prepared as a solution in 40%Cremophor-H₂O and administered at a dose of 150 μmol/kg p.o. The ratswere fasted for 24 hours before dosing. The efficiency of iron excretioninduced by 1 was 2.4±0.92%.

EXAMPLE 4 In Monkeys

Given the results in the rat model, the ability of 1 to promote ironexcretion in the iron-overloaded primate model [Blood, Vol. 79, Bergeronet al, “A Comparison of the Iron-Clearing Properties of1,2-Dimethyl-3-Hydroxypyrid-4-One, 1,2-Diethyl-3-Hydroxypyrid-4-One andDeferoxamine,” pages 1882-1890 (1992)] was evaluated. The drug wasprepared as a solution in 40% Cremophor-H₂O and administered at a doseof 150 μmol/kg p.o. The monkeys were fasted for 24 hours before dosing.Immediately prior to drug administration, the monkeys were sedated withketamine (7-10 mg/kg. i.m.) and given scopolamine (0.04-0.07 mg/kg/i.m.to prevent ketamine-related salivation and vomiting. At the dose of 150μmol/kg, the efficiency of 1 was 4.2±1.4% (n=4),

1. A method of preparing a compound of the formula:

wherein: Z is CH or N; R is H or acyl; R₁, R₂, R₃ and R₅ may be the sameor different and represent H, alkyl or hydrocarbyl arylalkyl having upto 14 carbon atoms; R₄ is H or alkyl having 1-4 carbon atoms, and saltsthereof with a pharmaceutically acceptable acid, comprising reacting afirst reagent of the formula:

with a second reagent of the formula:

or a salt thereof with a pharmaceutically acceptable acid.
 2. The methodof claim 1, wherein the first and second reagents are reacted in aninert solvent or a mixture of inert solvents.
 3. The method of claim 2,wherein the inert solvent or the mixture is aqueous.
 4. The method ofclaim 2, wherein the mixture comprises an aqueous solvent and analcohol.
 5. The method of claim 4, wherein the aqueous solvent is aphosphate buffer.
 6. The method of claim 4, wherein the alcohol ismethanol.
 7. The method of claim 4, wherein the mixture comprises aphosphate buffer and methanol.
 8. The method of claim 2, wherein thefirst and second reagents are reacted at ambient temperature.
 9. Themethod of claim 2, wherein the first and second reagents are reacted ata temperature from about ambient temperature to about 80° C.
 10. Themethod of claim 9, wherein the temperature is from about 50° C. to about80° C.
 11. The method of claim 10, wherein the temperature is about 70°C.
 12. The method of claim 2, wherein the first and second reagents arereacted under an inert gas atmosphere.
 13. The method of claim 1,wherein the first and second reagents are reacted in the presence of abase.
 14. The method of claim 13, wherein the base is NaHCO₃.
 15. Themethod of claim 1, wherein Z is CH.
 16. The method of claim 15, whereinR═R₁═R₂═R₄═R₅═H.
 17. The method of claim 1, wherein Z is N.
 18. Themethod of claim 1, wherein Z is CH and R₃ is an alkyl having 1-4 carbonatoms.
 19. A method of preparing a compound of the formula:

wherein: Z is CH or N; R₁, R₂, R₃ and R₅ may be the same or differentand represent H, alkyl or hydrocarbyl arylalkyl having up to 14 carbonatoms; and salts thereof with a pharmaceutically acceptable acid,comprising: a) reacting a first reagent of the formula:

wherein the hydroxyl groups of the first reagent are optionallyprotected by protecting groups, with a second reagent of the formula:

or a salt thereof with a pharmaceutically acceptable acid; and b)removing the protecting groups, if present.
 20. The method of claim 19,wherein the first and second reagents are reacted in an inert solvent ora mixture of inert solvents.
 21. The method of claim 20, wherein theinert solvent or the mixture is aqueous.
 22. The method of claim 21,wherein the mixture comprises an aqueous solvent and an alcohol.
 23. Themethod of claim 22, wherein the aqueous solvent is a phosphate buffer.24. The method of claim 22, wherein the alcohol is methanol.
 25. Themethod of claim 22, wherein the mixture comprises a phosphate buffer andmethanol.
 26. The method of claim 20, wherein the first and secondreagents are reacted at ambient temperature.
 27. The method of claim 20,wherein the first and second reagents are reacted at a temperature fromabout ambient temperature to about 80° C.
 28. The method of claim 27,wherein the temperature is from about 50° C. to about 80° C.
 29. Themethod of claim 28, wherein the temperature is about 70° C.
 30. Themethod of claim 20, wherein the first and second reagents are reactedunder an inert gas atmosphere.
 31. The method of claim 19, wherein thefirst and second reagents are reacted in the presence of a base.
 32. Themethod of claim 31, wherein the base is NaHCO₃.
 33. The method of claim19, wherein Z is CH.
 34. The method of claim 33, wherein R₁═R₂═R₄═R₅═H.35. The method of claim 19, wherein Z is N.
 36. The method of claim 19,wherein Z is CH and R₃ is an alkyl having 1-4 carbon atoms.
 37. Themethod of claim 19, wherein the protecting groups are removed bysolvolysis.
 38. The method of claim 19, wherein the protecting groupsare removed by reduction.
 39. A method of preparing a compound of theformula:

and salts thereof with a pharmaceutically acceptable acid, comprising:a) reacting a first reagent of the formula:

wherein the hydroxyl groups of the first reagent are optionallyprotected by protecting groups, with a second reagent of the formula:

or a salt thereof with a pharmaceutically acceptable acid; and b)removing the protecting groups, if present.
 40. The method of claim 39,wherein the first and second reagents are reacted in an inert solvent ora mixture of inert solvents.
 41. The method of claim 40, wherein theinert solvent or the mixture is aqueous.
 42. The method of claim 41,wherein the mixture comprises an aqueous solvent and an alcohol.
 43. Themethod of claim 42, wherein the aqueous solvent is a phosphate buffer.44. The method of claim 42, wherein the alcohol is methanol.
 45. Themethod of claim 42, wherein the mixture comprises a phosphate buffer andmethanol.
 46. The method of claim 40, wherein the first and secondreagents are reacted at ambient temperature.
 47. The method of claim 40,wherein the first and second reagents are reacted at a temperature fromabout ambient temperature to about 80° C.
 48. The method of claim 47,wherein the temperature is from about 50° C. to about 80° C.
 49. Themethod of claim 48, wherein the temperature is about 70° C.
 50. Themethod of claim 40, wherein the first and second reagents are reactedunder an inert gas atmosphere.
 51. The method of claim 39, wherein thefirst and second reagents are reacted in the presence of a base.
 52. Themethod of claim 51, wherein the base is NaHCO₃.
 53. The method of claim39, wherein the protecting groups are removed by solvolysis.
 54. Themethod of claim 39, wherein the protecting groups are removed byreduction.